NextSeq 500 sequencing; 2-hydroxyglutarate modulates histone methylation using both RNA/ChIP-seq
2-hydroxyglutarate modulates histone methylation using both RNA/ChIP-seq
ChIP:  cells were collected alongside with the media, and the DNA/histones crosslinking has been achieved by incubation for 10 minutes   with formaldehyde 1% final concentration. The reaction was stopped with the addition of glycine (166 mM final concentration) and incubation for extra 10 minutes.  ChIP: The cells have been washed one time with PBS and two times with PBS containing proteinase inhibitors (Complete Protease Inhibitor Cocktail, Merk 11697498001).  To lyse  the cells, SDS lysis buffer (1% SDS, 10 mM EDTA, 80 mM Tris-HCl pH?) was used with the addition of proteinase inhibitors. The cells were firstly incubated for 12 minutes at room temperature followed by the addition of 0.5 mm glass beads (Sigma Aldrich, 18406) and processing with the Precellys 24 homogenizer (Bertin Technologies) for 3 cycles of 30 seconds on and 30 seconds off at max speed and 10-16oC . The DNA was fragmented using the Bioruptor Pico sonicator (Diagenode) set to sonicate at 8oC for 45 cycles, 30 seconds followed by 30 seconds pause. H3K4me3 was targeted with Merk-Millipore,17-614, H3K36me3 was targeted with Abcam, ab9050, H3K36me2 was targeted with Abcam, ab9049. ChIP: TruSeq unstranded mRNA Library Prep kit (Illumina) was used to prepare libraries